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References (5)

Title : Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax - Cohen_2000_Infect.Immun_68_4549
Author(s) : Cohen S , Mendelson I , Altboum Z , Kobiler D , Elhanany E , Bino T , Leitner M , Inbar I , Rosenberg H , Gozes Y , Barak R , Fisher M , Kronman C , Velan B , Shafferman A
Ref : Infect Immun , 68 :4549 , 2000
Abstract : Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.
ESTHER : Cohen_2000_Infect.Immun_68_4549
PubMedSearch : Cohen_2000_Infect.Immun_68_4549
PubMedID: 10899854

Title : Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding - Shafferman_1992_J.Biol.Chem_267_17640
Author(s) : Shafferman A , Kronman C , Flashner Y , Leitner M , Grosfeld H , Ordentlich A , Gozes Y , Cohen S , Ariel N , Barak D , Harel M , Silman I , Sussman JL , Velan B
Ref : Journal of Biological Chemistry , 267 :17640 , 1992
Abstract : Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
ESTHER : Shafferman_1992_J.Biol.Chem_267_17640
PubMedSearch : Shafferman_1992_J.Biol.Chem_267_17640
PubMedID: 1517212

Title : Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit - Kronman_1992_Gene_121_295
Author(s) : Kronman C , Velan B , Gozes Y , Leitner M , Flashner Y , Lazar A , Marcus D , Sery T , Papier Y , Grosfeld H , Cohen S , Shafferman A
Ref : Gene , 121 :295 , 1992
Abstract : To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
ESTHER : Kronman_1992_Gene_121_295
PubMedSearch : Kronman_1992_Gene_121_295
PubMedID: 1446827

Title : The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant - Velan_1991_J.Biol.Chem_266_23977
Author(s) : Velan B , Grosfeld H , Kronman C , Leitner M , Gozes Y , Lazar A , Flashner Y , Marcus D , Cohen S , Shafferman A
Ref : Journal of Biological Chemistry , 266 :23977 , 1991
Abstract : Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.
ESTHER : Velan_1991_J.Biol.Chem_266_23977
PubMedSearch : Velan_1991_J.Biol.Chem_266_23977
PubMedID: 1748670

Title : Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme - Velan_1991_Cell.Mol.Neurobiol_11_143
Author(s) : Velan B , Kronman C , Grosfeld H , Leitner M , Gozes Y , Flashner Y , Sery T , Cohen S , Ben-Aziz R , Seidman S , Shafferman A , Soreq H
Ref : Cellular Molecular Neurobiology , 11 :143 , 1991
Abstract : 1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.
ESTHER : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedSearch : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedID: 1849451